Programmed cell death is a natural process by which an organism eliminates particular cells in a regulated process. Programmed cell death pathways are initiated by a variety of stimuli, some internal to the cell (e.g. DNA damage) while others are activated as a result of external factors (e.g. fas ligand). To date, a single mediator of apoptosis has not been elucidated. Rather, a variety of apoptotic mechanisms are in a potential state for response at all times. The fact that many molecules are identified as possessing both pro- and anti-apoptotic activity (e.g. caspases, NF-kB, AP1, and p53), there appears to be a fine balance of factors which permit survival or terminate the cell.
While no single mediator of apoptosis has been identified, mutations in p53 are recognized as the most common genetic alterations associated with human cancer, diseases characterized by abberent regulation of cell cycle control and apoptosis. Although p53 was first characterized as a dominant cooperating oncogene, it was later determined that the form of p53 under investigation at that time was a mutant, inactive and long-lived form of the wild-type protein. This mutant protein displaced the short-lived wild-type native form from its enzymatic substrate binding sites and was mistakenly deemed to act dominantly. p53 is now thought of as a tumor suppressor gene.
The sequence specific DNA binding properties of p53 regulate the transcription of a continually expanding number of genes involved in regulation of the cell cycle and apoptosis. p53 has been implicated in the transcriptional downregulation of the bcI2 gene and upregulation of the bax gene, precipitating a cascade of events leading to apoptosis. The complete mechanism of action of p53 is not fully elucidated. Though it is known that p53 binds to many important cellular proteins and is involved in the control of gene expression, cell cycle regulation and apoptosis, it is observed that some tumors produce wildtype p53, yet do not undergo apoptosis. The intracellular cytosolic concentration of p53 appears to be maintained by a balance of continual synthesis and degradation. p53 exists as an inactive cytosolic monomeric protein with a very short half-life. Increasing data support the view that, in normal cells, stability of p53 is controlled through mdm-2-binding and ubiquination, through an, as yet, incompletely understood mechanism. Binding of p53 to mdm-2 has been shown to lead to ubiquitination of p53 protein. Following ubiquitination, the p53 is believed to be degraded through a common proteasome mediated degradative pathway. p53 exerts its function as a tetramer of phosphorylated p53 subunits. The phosphorylation step leading to activation of p53 function is mediated by DNA-dependent protein kinase (“DNA-PK”). DNA-PK appears to be activated in response to DNA damage. Woo, et al. (1998) Nature 394:700–704; Lane, D. (1998) Nature 394:616–617.
While not the sole mechanism of apoptosis, the p53 apoptotic pathway is clearly an important pathway employed by the organism to eliminate aberrant cell types. p53 has been demonstrated to play a role in many biochemical pathways related to human carcinogenesis. In approximately 50% of human cancers, the p53 gene is inactivated through mutational, viral, or other cellular components. Somatic mutations in the DNA binding domain of p53 are found in the majority of human tumors bearing p53 alterations. It has been demonstrated that restoration of p53 function in p53 deficient tumor cells will induce apoptosis of tumor cells. Currently, replication defective recombinant adenovirus vectors expressing wildtype p53 (rAd-p53) are being used in human clinical trials (Nielsen and Maneval, 1998). To date, more than 137 human patients have been treated with rAd-p53. Initial results of these trials demonstrate acceptable safety profies and therapeutic effect of such vectors in vivo.
Calpain, a calcium dependent cysteine protease, has recently been implicated to play a role in apoptosis. Calpain has two primary isoforms (m-calpain and μ-calpain) distinguished by the calcium concentration required for their activation in vitro. Calpain consists of a heavy and light chain. The heavy chain consists of a cysteine proteinase domain. The light chain is a calcium binding domain and possesses four EF-hands characteristic of calcium binding proteins. The native form of calpain is inactive except at relatively high Ca+2 concentrations. In vitro experiments indicate that μ-calpain (also known as calpain I) requires μM calcium levels for activation. In contrast, m-calpain (also known as calpain II) requires a much higher (mM) calcium concentration for activation. Calpain is a cytosolic protease whose activity is modulated by the cytosolic inhibitor protein, calpastatin. Upon activation, calpain has been shown to translocate to the cell membrane where it is sequestered from its inhibitors (Lane et al., 1992; Molinari and Carafoli, 1997).
Additionally, calpain has been shown to possess in vitro proteolytic action against a broad range of substrates including components of receptor signaling pathways, interleukin receptors where the common cytokine receptor γ chain is cleaved by calpains (Noguchi, et al. (1997) Proc Natl Acad Sci USA 94, 11534–9), cytoskeletal and focal adhesion proteins (Schoenwaelder, et al. (1997) J. Biol. Chem 272: 1694–702.), integrins (Du, et al. (1995) J Biol Chem 270, 26146–51; Inomata, et al. (1996) Arch Biochem Biophys 328, 129–34.; Palecek, et al. (1998) J. Cell Sci. 111, 929–40), and transcriptional factors such as c-fos and c-jun (Jariel-Encontre, I., Salvat, C., Steff, A. M., Pariat, M., Acquaviva, C., Furstoss, O., and Piechaczyk, M. (1997) Mol Biol Rep 24, 51–6.; Suzuki, K., Saido, T. C., and Hirai, S. (1992) Ann N Y Acad Sci 674, 218–27.), NF-kappa B (Claudio, et al. (1996) Exp Cell Res 224, 63–71.; Liu, et al. (1996) FEBS Lett 385, 109–13.), NF2 (Kimura, et al. (1998) Nature Medicine 4:915–922) and p53 (Gonen, et al. (1997) FEBS Lett 406, 17–22.; Kubbutat, M. H., and Vousden, K. H. (1997) Mol Cell Biol 17, 460–8.).
Although calpains demonstrate a broad range of substrates in vitro, its role in vivo remains unclear. As a consequence of their broad substrate specificity, the therapeutic applications suggested for calpain inhibitors include the prevention of neurodegradations and cellular damage caused by excessive activation of glutamate receptors in neuronal cells, retarding cataract formation, minimization of degeneration of neuronal tissues following injury, mycoardial infaction, angioplastic restenosis, prevention of cartilage damage and subsequent inflammation, muscular dystrophy and platelet aggregation associated with thrombosis. In vivo experiments in mice and rats have shown that prior infusion of calpain inhibitors prevents neuronal and hepatocyte cell death due to hypoxia, and increases survival after orthotopic liver transplants. In vitro, calpain inhibitors have been observed to attenuate apoptosis in primary rat hepatocytes in response to a variety of apoptotic stimuli. Additionally, calpain inhibitors have been shown to inhibit activation-induced programmed cell death of T-cells from HIV positive donors (Sarin et al., 1994). Calpain has been shown to cleave the precursor form of IL-1a into a 17 kD C-terminal fragment referred to as “mature” IL-1a and a 16 kD N-terminal fragment which migrates to the nucleus and induces apoptosis in vitro. In each of these instances, the primary application of the calpain inhibitor is to prevent cell death.
p53 gene therapy has focused on the use of recombinant viral, particularly adenoviral, vectors to result in the intracellular expression of p53. In such therapeutic regimens, a significant excess of the recombinant virus must be administered to the patient because not every viral particle administered will infect the target cell. This is due at least in part to the effects of diffusion, clearance and/or neutralization by the organism, and that not every interaction between the viral particle and a target cell results in infection and productive expression. However, the systemic administration of excess recombinant virus poses potential safety concerns to the individual undergoing treatment. Consequently, methods which can be employed to lower the total dose of the recombinant viral vector while maintaining a therapeutically effective dose are desired. The present invention provides a method of inducing apoptosis in p53 positive tumor cells by the administration of a calpain inhibitor alone or in combination with rAd-p53 or in combination with rAd-p53 and in tumors with mutated or null p53 status. The present invention also provides a decreased CTL response to the administration of recombinant adenoviral vectors by the concomitant administration of calpain inhibitors. The combined effect being increased p53 expression.